Influence of Companion Planting on Microbial Compositions and Their Symbiotic Network in Pepper Continuous Cropping Soil

Continuous cropping obstacles have become a serious factor restricting sustainable development in modern agriculture, while companion planting is one of the most common and effective methods for solving this problem. Here, we monitored the effects of companion planting on soil fertility and the microbial community distribution pattern in pepper monoculture and companion plantings. Soil microbial communities were analyzed using high-throughput sequencing technology. Companion plants included garlic (T1), oat (T2), cabbage (T3), celery (T4), and white clover (T5). The results showed that compared with the monoculture system, companion planting significantly increased the activities of soil urease (except for T5) and sucrase, but decreased catalase activity. In addition, T2 significantly improved microbial diversity (Shannon index) while T1 resulted in a decrease of bacterial OTUs and an increase of fungal OTUs. Companion planting also significantly changed soil microbial community structures and compositions. Correlation analysis showed that soil enzyme activities were closely correlated with bacterial and fungal community structures. Moreover, the companion system weakened the complexity of microbial networks. These findings indicated that companion plants can provide nutrition to microbes and weaken the competition among them, which offers a theoretical basis and data for further research into methods for reducing continuous cropping obstacles in agriculture.


Microbial Community Analysis
Analysis of the raw data was conducted using FASTP (V 0.18.0), including read filtering and splicing, [29] and FLASH (version 1.2.11) [30]. QIIME (version 1.9.1) [31] was used to filter low-quality tags to obtain high-quality clean tags for subsequent analysis. The 16S rRNA and ITS gene sequences were clustered within operational taxonomic units (OTUs) at a 97% similarity level using UPARSE (version 9.2.64) [32]. The Ribosomal Database Project (RDP) Classifier (http://rdp.cme.msu.edu/) was used to analyze the taxonomy of each bacterial and fungal gene sequence against the Silva database (version 132) and Unite database (version 8.0) with a confidence threshold of 80% [33,34]. A constrained principal coordinate analysis (PCoA) was performed using weighted UniFrac distances based on the OTU level. The bacterial and fungal community compositions were visualized using Circos software (http://circos.ca/). The linear discriminant analysis effect size (LEfSe) method was used to identify the most differentially abundant biomarker taxa among groups [35]. Alpha-diversity indices were calculated by QIIME [31]. To characterize the effects of companion planting on soil microbial co-occurrence patterns, a Spearman correlation matrix among soil bacterial and fungal genera was calculated using the "hmisc" and "igraph" packages. Strong correlations of bacterial and fungal genera with relative abundances greater than 0.1% (R > 0.7, p < 0.05) were retained for the visualization of networks using Gephi software [36][37][38]. Genera with the highest betweenness centrality scores were considered keystone taxa (top five genera), which have a unique and crucial role in microbial community and their deficiency can cause dramatic shifts in microbial structure and function.

Statistical Analysis
One-way analysis of variance (ANOVA) with Duncan's test was selected to analyze the differences in soil enzyme activities and α-diversity indices among all treatments. Analysis of similarity (ANOSIM) was performed to test whether the differences between groups were significantly greater than that within groups. Redundancy analysis (RDA) was performed to present the correlations between soil enzymes and microbial community structures using the vegan package in R with 999 permutations. Spearman correlation analysis was performed using IBM SPSS Statistics (v 25.0) (SPSS, USA). A p-value of < 0.05 was considered statistically significant.

Influence of Companion Planting on Soil Enzyme Activities
The soil enzyme activities of pepper in monoculture and five companion systems are shown in Table 1. Compared with pepper monoculture, companion planting significantly increased soil urease (except for T5) and sucrase activities (p < 0.05), while soil catalase activity was significantly reduced (p < 0.05), and no significant differences were observed in the CK and T3 treatments.

Soil Microbial Diversity and Community Structures
A total of 3,435,240 16S rRNA and 3,690,816 ITS quality sequences were obtained from all monoculture and companion soil samples, and the sequences were grouped into 4,825 bacterial and 542 fungal OTUs. The rarefaction curves (Fig. S1) for all samples indicated that the sequencing depth has reached an extent that covered all species.
A petal plot was used to count the number of common and unique OTUs in all groups. As shown in Fig. 1, the numbers of common bacterial and fungal OTUs were 2,750 and 280, respectively, and the number of unique bacterial OTUs in the companion system was less than that in the monoculture system, while the result trends in The mean value ± SD (n = 6). Different letters in the same column represent significant differences at the p = 0.05 level.
the number for unique fungal OTUs were opposite. The soil bacterial and fungal richness and diversity are shown in Table 2. The number of soil bacterial OTUs was significantly lower in the T1 treatment than that in the control, while T2 significantly increased the bacterial Shannon index (p < 0.05). For fungi, the T1 and T3 treatments significantly increased the number of OTUs, and T2 treatment resulted in an increase in the Shannon index  The mean value ± SD (n = 6). Different letters in the same column represent significant differences at the p = 0.05 level. compared to CK ( Table 2). The correlation analysis showed that the soil bacterial Shannon indices had no significant correlation with soil enzyme activities (p > 0.05), while fungal observed OTUs (r = 0.64, p =0.000; R = 0.55, p = 0.001) and Shannon indices (r = 0.43, p = 0.008; R = 0.52, p = 0.001) had significant positive correlations with urease and sucrase activities (Table 3).
PCoA was conducted to elucidate the shifts in the soil bacterial and fungal community structures in six treatments. PCoA plots revealed clear distinction in bacterial and fungal communities based on the OTUs level ( Fig. 2, Table S1). The results showed that companion planting significantly changed soil microbial community structures, and there were significant differences among all treatments (except for T4-T5 fungi). The RDA showed that soil urease, catalase and sucrase activities were significantly correlated with bacterial and fungal community structures (p < 0.001) (Fig. 3, Table S2).

Relative Abundances of Major Bacterial and Fungal Taxa
Companion planting treatments caused changes in the soil bacterial and fungal community compositions. The Circos plots show the composition and abundances of microbial communities at the phylum and genus levels in soil (Fig. 4, Tables S3 and S4). The top 10 bacterial phyla in all soil samples were Proteobacteria, Acidobacteria, Planctomycetes, Gemmatimonadetes, Actinobacteria, Bacteroidetes, Chloroflexi, Verrucomicrobia, Patescibacteria, and Firmicutes (Fig. 4A, Table S3), with relative abundances averaging 36.57, 17.04, 12.04, 7.83, 7.11, 5.55, 5.24, 2.23, 1.64, and 1.26%, respectively, and these 10 phyla accounted for 96.50% of all sequences. Companion planting treatments increased the relative abundances of Planctomycetes and Gemmatimonadetes, and decreased those of Proteobacteria, Bacteroidetes and Chloroflexi. The T1, T2 and T3 treatments caused the increases in the abundances of Actinobacteria and Paesciabacteria, which were reduced by the T4 and T5 treatments, while the abundance of Firmicutes in all treatments followed the opposite pattern compared to CK. In addition, there were no significant differences in the relative abundance of Verrucomicrobia in any treatment (p > 0.05). The taxonomical classification showed that 137 bacterial genera were detected at the genus level. Sphingomonas, RB41, Lysobacter, Dongia, MND1, Acidibacter, Pseudoxanthomonas, Arenimonas, Altererythrobacter, and Ellin6055 were found to be the top 10 dominant bacterial genera (Fig. 4B, Table S3). Among the top 10 bacterial genera, companion planting treatments significantly increased the relative abundances of MND1 and Ellin6055, and decreased those of Lysobacter, Dongia, Acidibacter, Pseudoxanthomonas, and Arenimonas compared to CK. Except for the T1 treatment, other companion planting treatments significantly reduced the relative abundance of Sphingomonas, while that of Alterythrobacter was significantly increased by the companion planting treatments (except for T5). Only the relative abundance of RB41 had no significant difference in all treatments. In addition, 45 bacterial biomarkers were identified at the phylum, class, order, family, genus and species levels (LDA(log10) score > 3.5) (Fig. S3).
The fungal composition was dominated by Ascomycota, accounting for 93.23% of all sequences on average (Fig. 4C, Table S4). The subdominant phyla were Basidiomycota, Mortierellomycota, Chytridiomycota, Mucoromycota, and Glomeromycota. Significant differences were observed in the relative abundances of Ascomycota, Basidiomycota, Mortierellomycota, and Glomeromycota. Compared with monoculture, all companion treatments increased the relative abundances of Basidiomycota and Glomeromycota, and decreased that of Ascomycota. In addition, there were no significant differences in the abundances of Mortierellomycota, Chytridiomycota, and Mucoromycota. At the fungal genus level, Kotlabaea, Cladorrhinum, Coprinellus, Madurella, Schizothecium, Tetracladium, Pseudogymnoascus, Podospora, Scedosporium, and Mortierella were the top 10 dominant fungal genera (Fig. 4D, Table S4). Among the 10 genera, compared to monoculture, T4 significantly increased the relative abundances of Kotlabaea, Madurella, and Tetracladium, while T5 treatment increased those of Coprinellus, Schizothecium, and Podospora. T1 and T2 significantly increased the relative abundance of Pseudogymnoascus, but decreased that of Podospora. There were no significant differences in the abundances of Scedosporium and Mortierella. In addition, 98 biomarkers were found at the phylum, class, order, family, genus and species levels (with LDA(log10) score > 3.5) (Fig. S4).

Co-Occurrence Network of the Soil Microbial Community
Microbial co-occurrence network analysis was conducted to explore the co-occurrence patterns among bacteria and fungi at the genus level in soil. As shown in Fig. 5, 119 nodes and 544 edges were observed in the monoculture system (CK) network (R > 0.7, p < 0.05). Compared to CK, the numbers of nodes and edges in the five companion planting networks decreased, indicating that companion planting reduced the relationships among microorganisms, thus weakening the symbiotic patterns of the microbial community. In addition, the average degrees and clustering coefficients in the T3 and T5 treatments were lower than those of the control (CK), while the opposite results were observed in the T4 network. The network diameter in all companion treatments decreased compared to CK. Moreover, significant differences were also observed in the topological structure characteristics of the symbiosis network between control and companion planting treatments. Overall, companion planting treatments affected the network complexity of the microbial community. Additionally, there were differences in the keystone taxa in the microbial networks between the control and five companion planting treatments (Fig. 5). There were also significant differences in the positive and negative edges in the networks.

Discussion
Soil biological activity, referring to the transformation, release, and fixation of soil nutrients, is an important indicator to assess soil fertility and health status [39]. However, plant diversity also has a great impact on the soil environment. This study revealed the impact of companion planting on soil biological activity.
Soil enzymes are involved in soil biochemical processes and play key roles in the occurrence, transformation, and availability of soil nutrients. They can reflect soil biological activity and biochemical reaction intensity, and are also sensitive to external environmental changes. Moreover, soil enzyme activities are important biological indicators of soil fertility, quality and health [40]; for example, they can transform complex soil organic matter into nutrients that can be directly used by plants and microorganisms [41]. Previous studies have shown that a reasonable intercropping mode can improve soil enzyme activities to varying degrees, which is conducive to the accumulation and transformation of soil nutrients [42]. The reason why plant diversity affects enzyme activities is that microorganisms have the potential to mineralize or fix essential nutrients by releasing enzymes into soil solution, and increase or reduce their availability to crops [42]. Soil catalase sensitively reflects the intensity of soil microbiological processes and crop metabolic processes to a certain extent [43]. Some studies have shown that soil catalase activity is closely related to root respiration intensity, and high root respiration intensity is an important factor for increasing the catalase activity [44]. In this study, catalase activity were significantly decreased by companion planting treatments, which can be attributed to the fact that companion plants did not develop pepper roots. After companion planting with pepper, root density and respiration intensity decreased, which inhibited the production of hydrogen peroxide. This result differs from the results of many companion planting studies and may have been caused by a lack of companion plants, and the companion period was shorter in our study, which was related to soil pH. Enzyme activity is directly affected by pH, and most soil enzymes show maximum activity at slightly acidic pH values [45]. Sucrase, also known as invertase, is a key enzyme of soil carbon metabolism, and its enzymatic reaction produces glucose, so in addition to being a nutrient source for plants and microorganisms it can also increase soluble nutrients in soil [46]. We found that companion treatments significantly increased soil sucrase activity, which promoted the metabolism of plants and microorganisms and further improved plant growth. Soil urease mainly comes from soil microorganisms that can promote the transformation of soil nitrogen into inorganic ammonia and carbonic acid, and its activity directly reflects soil nitrogen supply capacity [47]. In our study, except for T5 treatment, other companion planting treatments significantly increased urease activity, indicating that plants can promote microbial nitrogen metabolism and provide important nutrients for plants. This may have been due to the activation of rhizosphere microorganisms by microbial interactions and the resulting improvement of soil enzyme activities, which caused changes in soil nutrient contents that sped up the growth of plants.
Soil microbial diversity plays an important role in maintaining multiple functions in terrestrial ecosystems [48], and the increases in soil microbial diversity is conducive to soil function and health [49]. Several studies have noted that management measures to increase plant diversity often affect soil microbial communities [42,50]. In this study, soil microbial diversity indices in companion planting were generally higher than that of monoculture. In addition, these indices were significantly different among the different treatments (Table 2), which is consistent with previous studies [51]. The improvement of soil microbial diversity by companion planting may be the reason why companion plants can increase pepper biomass [52]. Similar results were found in other studies, which also indicated that companion planting increased the microbial diversity of pepper continuous cropping soil and changed the soil microbial community structures. Companion planting systems play an important roles in the soil microbial diversity and community composition [53], and the reason may be due to the microbial interactions. In addition, the beta diversity showed that there were significant differences in microbial structures and compositions among different companion planting treatments (Fig. 2, Table S1), which may be caused by root exudates secreted by different plants. Similar results have been reported in previous soil microbial studies [54]. These results further confirmed the complexity from a micro perspective.
Intercropping has been shown to change soil microbial community compositions [55]. Changes in bacterial and fungal communities were mostly accompanied by differences in the abundances of specific phyla. In the present study, at the bacterial phylum level, the dominant bacterial taxa included Proteobacteria, Acidobacteria, Planctomycetes, Gemmatimonadetes, Actinobacteria, Bacteroidetes, Chloroflexi, Verrucomicrobia, Patescibacteria, and Firmicutes (Fig. 4A), suggesting that these microbial communities have high adaptability and play an important role in these ecosystems [56]. However, it was found that Proteobacteria was the dominant phylum in most studies [57,58], which is the most abundant bacterial phylum in terrestrial habitats and is positively related to C mineralization [56]. In this study, companion planting led to increased relative abundances of Planctomycetes and Gemmatimonadetes and decreased those of Proteobacteria and Chloroflexi, which was inconsistent with the results of previous intercropping studies [57]. The reasons may be related to different soil properties and cultivation methods, and further study is needed. Proteobacteria feed on different stubborn carbon sources and strongly impact soil microbial community structures by providing functional capacity for the carbon cycle and more available nutrients for plant growth [59,60]. As an important microorganism in the environment, Planctomycetes plays an important role in the biogeochemical cycle, such as in the carbon and nitrogen cycles [61,62]. Our study found that companion planting significantly increased the relative abundance of Planctomycetes, indicating that plants can promote the metabolic cycle of main crops and stimulate plant growth. Therefore, the identification of this phylum may reflect the difference in the nitrogen fixation effect among different groups, indicating that companion planting may be an important way to improve the nitrogen fixation effect [4]. Ascomycota, Basidiomycota, Glomeromycota, and Mortierellomycota were detected as the dominant fungal phyla across all soil samples, in agreement with previous results [63]. Ascomycota, the most abundant group in the fungal phylum, is widely distributed in soil all over the world and can adapt to a variety of environments [64]. In addition, companion planting clearly shifted soil bacterial and fungal communities at the genus level, which is consistent with the previous research results, indicating that companion planting has an overall impact on microbial community composition. Pathogenic Fusarium was not the dominant fungus in this study, indicating that companion planting improves soil resistance and reduces its pathogenicity [65]. These results are of great significance to soil health status. To further understand the impact of the process mechanism of companion planting on pepper continuous cropping soil fertility and health, further basic research on functional genes is needed [56]. Meanwhile, our analysis of microbial diversity showed that there were differences in nitrogen fixation and other related microorganisms in different groups. Therefore, nutrient cycling and its microbial community are the biological basis for the improvements provided by companion planting [4].
Network analysis can be used to determine the interactions of microbial taxa in a niche and the keystone species that have the greatest impact on the microbial community [66]. A symbiotic network is an important factor affecting the stability of the microbial community under external interference. In the microbial networks obtained, the number of nodes and edges between bacterial and fungal genera both decreased in all of the companion systems compared to the monoculture system (Fig. 5), indicating that companion planting can reduce the competition between microorganisms, which may be because plant diversity improves nutrient availability and cycling. However, Pivato et al. [67] found that the effect of pea/wheat intercropping on rhizosphere bacterial network was not significant, which may be caused by the different planting methods as there are some differences between intercropping and companion planting. As one of the most common and effective planting methods, companion planting may have the ability to affect the symbiosis mode of soil microorganisms, and change it to a response mode of external interference. In this process, some synergistic microbial symbiosis modes may be generated to promote symbiosis and synergy among soil microorganisms, so further research in this area is needed.
In this study, RDA plots showed that soil enzyme activities were significantly correlated with soil microbial community structures, including urease, catalase, and sucrase (Fig. 3). In addition to enzyme activities, many studies have found that environmental factors such as pH, soil organic carbon, and available phosphorus determine soil microbial communities in different ecosystems [52,68,69]. The bacterial and fungal community structures were positively correlated with organic carbon, which can improve the soil environment by increasing soil carbon and reducing soil acidification to affect soil microbial communities. However, in companion planting systems, the environmental factors affecting soil microbial community structures need to be further studied.
To sum up, soil microbial community distribution patterns of pepper monoculture and companion plantings were compared using high-throughput sequencing technology. The results showed that companion planting significantly increased soil urease and sucrase activities, but decreased catalase activity compared to the monoculture system. In addition, companion planting improved the microbial diversity in pepper continuous cropping soil, and significantly changed soil microbial community structures and compositions. Soil enzyme activities were also found to be significantly correlated with microbial community structures. Moreover, the companion system reduced the complexity of microbial community networks, which may result in the provision of microbial nutrition and weaken the competition among microorganisms. These results should provide a theoretical basis and data for further research on reducing continuous cropping obstacles in agriculture.